Dolores Gonzalez Pacanowska

Novel Azasterols as Potential Agents for Treatment of Leishmaniasis and Trypanosomiasis

ABSTRACT This paper describes the design and evaluation of novel azasterols as potential compounds for the treatment of leishmaniasis and other diseases caused by trypanosomatid parasites. Azasterols are a known class of (S)-adenosyl-l-methionine: Δ24-sterol methyltransferase(24-SMT) inhibitors in fungi, plants, and some parasitic protozoa. The compounds prepared showed activity at micromolar and nanomolar concentrations when tested against Leishmania spp. and Trypanosoma spp. The enzymatic and sterol composition studies indicated that the most active compounds acted by inhibiting 24-SMT. The role of the free hydroxyl group at position 3 of the sterol nucleus was also probed. When an acetate was attached to the 3β-OH, the compounds did not inhibit the enzyme but had an effect on parasite growth and the levels of sterols in the parasite, suggesting that the acetate group was removed in the organism. Thus, an acetate group on the 3β-OH may have application as a prodrug. However, there may be an additional mode(s) of action for these acetate derivatives. These compounds were shown to have ultrastructural effects on Leishmania amazonensis promastigote membranes, including the plasma membrane, the mitochondrial membrane, and the endoplasmic reticulum. The compounds were also found to be active against the bloodstream form (trypomastigotes) of Trypanosoma brucei rhodesiense, a causative agent of African trypanosomiasis.

Design, synthesis, and evaluation of inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper concerns the design, synthesis, and evaluation of inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Initially study was made of the structures of the leishmanial and human enzyme active sites to see if there were significant differences which could be exploited for selective drug design. Then a series of compounds were synthesized based on 5-benzyl-2, 4-diaminopyrimidines. These compounds were assayed against the protozoan and human enzymes and showed selectivity for the protozoan enzymes. The structural data was then used to rationalize the enzyme assay data. Compounds were also tested against the clinically relevant forms of the intact parasite. Activity was seen against the trypanosomes for a number of compounds. The compounds were in general less active against Leishmania. This latter result may be due to uptake problems. Two of the compounds also showed some in vivo activity in a model of African trypanosomiasis.

Phosphonosulfonates Are Potent, Selective Inhibitors of Dehydrosqualene Synthase and Staphyloxanthin Biosynthesis in Staphylococcus aureus

Staphylococcus aureus produces a golden carotenoid virulence factor called staphyloxanthin (STX), and we report here the inhibition of the enzyme, dehydrosqualene synthase (CrtM), responsible for the first committed step in STX biosynthesis. The most active compounds are halogen-substituted phosphonosulfonates, with K(i) values as low as 5 nM against the enzyme and IC(50) values for STX inhibition in S. aureus as low as 11 nM. There is, however, only a poor correlation (R(2) = 0.27) between enzyme and cell pIC(50) (= -log(10) IC(50)) values. The ability to predict cell from enzyme data improves considerably (to R(2) = 0.72) with addition of two more descriptors. We also investigated the activity of these compounds against human squalene synthase (SQS), as a counterscreen, finding several potent STX biosynthesis inhibitors with essentially no squalene synthase activity. These results open up the way to developing potent and selective inhibitors of an important virulence factor in S. aureus, a major human pathogen.

Novel Inhibitors of Leishmanial Dihydrofolate Reductase

The program DOCK3.5 was used to search the Cambridge Structural Database for novel inhibitors of Leishmanial dihydrofolate reductase. A number of compounds were obtained and screened against the enzyme and against the intact parasite Leishmania donovani and the related organisms Trypanosoma brucei and Trypanosoma cruzi. The compounds screened showed weak activity in both the enzyme assays and the in vitro assays.

New Azasterols against Trypanosoma brucei: Role of 24-Sterol Methyltransferase in Inhibitor Action

ABSTRACT A series of azasterol derivatives, designed as potential inhibitors of the Δ24-sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.

Design, synthesis and evaluation of novel uracil acetamide derivatives as potential inhibitors of Plasmodium falciparum dUTP nucleotidohydrolase.

The ubiquitous enzyme dUTP nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and can be considered as the first line of defence against incorporation of uracil into DNA. Inhibition of this enzyme results in over-incorporation of uracil into DNA, leading to DNA fragmentation and cell death and is therefore lethal. By taking advantage of structural differences between the human and Plasmodium dUTPase, selective inhibitors of the enzyme can be designed and synthesised with the aim of being developed into novel anti-parasitic drugs. Analogue based design was used to target the Plasmodium falciparum dUTPase (PfdUTPase). The structures of previously discovered selective inhibitors of the PfdUTPase were modified by insertion of an amide bond. A series of tritylated uracil acetamide derivatives were synthesised and assessed for inhibition of the enzyme and parasite growth in vitro. These compounds were weak inhibitors of the PfdUTPase.

The structure-based design and synthesis of selective inhibitors of Trypanosoma cruzi dihydrofolate reductase

This paper describes the design and synthesis of potential inhibitors of Trypanosoma cruzi dihydrofolate reductase using a structure-based approach. A model of the structure of the T. cruzi enzyme was compared with the structure of the human enzyme. The differences were used to design modifications of methotrexate to produce compounds which should be selective for the parasite enzyme. The derivatives of methotrexate were synthesised and tested against the enzyme and intact parasites.

Δ24 (25)-sterol methenyltransferase: Intracellular localization and azasterol sensitivity in Leishmania major promastigotes overexpressing the enzyme

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine:Delta(24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniases. Here, we report studies concerning the intracellular distribution of sterol methenyltransferase in Leishmania major promastigotes and overexpressing cells using a specific antibody raised against highly purified recombinant protein. It was found by immunofluorescence and electron microscopy studies that in L. major wild-type cells sterol methenyltransferase was primarily associated to the endoplasmic reticulum. In addition to this location, the protein was incorporated into translucent vesicles presumably of the endocytic pathway. We also found in this study that cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22, 26 azasterol.

Synthesis and testing of 5-benzyl-2,4-diaminopyrimidines as potential inhibitors of leishmanial and trypanosomal dihydrofolate reductase.

Dihydrofolate reductase is a drug target that has not been thoroughly investigated in leishmania and trypanosomes. Work has previously shown that 5-benzyl-2,4-diaminopyrimidines are selective inhibitors of the leishmanial and trypanosome enzymes. Modelling predicted that alkyl/aryl substitution on the 6-position of the pyrimidine ring should increase enzyme activity of 5-benzyl-2,4-diaminopyrimidines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Various compounds were prepared and evaluated against both the recombinant enzymes and the intact organisms. The presence of a substituent had a small or negative effect on activity against the enzyme or intact parasites compared to unsubstituted compounds.

Targeting kinetoplastid and apicomplexan thymidylate biosynthesis as an antiprotozoal strategy

Kinetoplastid and apicomplexan parasites comprise a group of protozoans responsible for human diseases, with a serious impact in human health and the socioeconomic growth of developing countries. Chemotherapy is the main option to control these pathogenic organisms and nucleotide metabolism is considered a promising area for the provision of antimicrobial therapeutic targets. Impairment of thymidylate (dTMP) biosynthesis severely diminishes the viability of parasitic protozoa and the absence of enzymatic activities specifically involved in the formation of dTMP (e.g. dUTPase, thymidylate synthase, dihydrofolate reductase or thymidine kinase) results in decreased deoxythymidine triphosphate (dTTP) levels and the so-called thymineless death. In this process the ratio of deoxyuridine triphosphate (dUTP) versus dTTP in the cellular nucleotide pool has a crucial role. A high dUTP/dTTP ratio leads to uracil misincorporation into DNA, the activation of DNA repair pathways, DNA fragmentation and eventually cell death. The essential character of dTMP synthesis has stimulated the interest in the identification and development of drugs that specifically block the biochemical steps involved in thymine nucleotide formation. Here, we review the available literature in relation to drug discovery studies targeting thymidylate biosynthesis in kinetoplastid (genera Trypanosoma and Leishmania) and apicomplexan (Plasmodium spp and Toxoplasma gondii) protozoans. The most relevant findings concerning novel inhibitory molecules with antiparasitic activity against these human pathogens are presented herein.